Although we are not prodigious, we may discover and understand important things through curiosity, good observation, and perseverance

Establishing a sustainable food supply is essential to cope with the rapidly increasing population and it will be one of the most significant challenges to human society in the near future. Therefore, food science research needs to focus on economic, sustainable, and safe production of high-quality food products from agricultural raw materials. In addition, it will be necessary to minimize the production of food wastes for developing efficient food production processes. Given these societal needs, the long-term goal of our research program is to develop microbial fermentation processes that utilize metabolic engineering approaches for producing food ingredients, value-added chemicals, and biofuels while minimizing the production of wastes.

To this end, our research group has been conducting microbial metabolic engineering research to enable efficient production of value-added products from inexpensive and abundant raw materials, such as non-edible plant cell walls and wastes from food manufacturing processes. The results obtained from our research have contributed to the industrial production of value-added products and a deeper understanding of cellular metabolism and its regulatory mechanisms for microbial strain improvement.

Microbial production of biofuels and value-added chemicals from plant cell wall. We have developed engineered yeast strains capable of producing ethanol from plant cell wall hydrolysates rapidly and efficiently. The results were published in respected journals in the field. Many related patent applications had also been filed. We have constructed engineered yeast strains that can produce not only biofuels, but also various value-added chemicals from the sugars prevalent in plant cell wall hydrolysates. For instance, we have constructed an engineered yeast strain capable of converting xylose into lactic acid, which can be used for making bio-based plastics. The resulting strain produced lactic acid with a high yield and productivity from xylose. In addition, we constructed many engineered yeast strains that can produce 2,3-butanediol used for producing synthetic rubber, isoprenoids used as food and medicine, and 3-hydroxybutryrate used for producing functional polymers. Our efforts will contribute to the transformation of a chemical industry that has relied on petroleum into a bio-based refinery industry for sustainable chemical production.

Metabolic engineering to overcome toxic inhibitors in plant cell wall hydrolysates. Plant cell wall hydrolysates contain large amounts of toxic chemicals inhibiting the growth and fermentation of microorganisms. As such, it is essential to develop engineered yeast strains capable of tolerating the toxic inhibitors in order to efficiently convert plant cell hydrolysates into biofuels and chemicals. We have devised a synthetic metabolic pathway which can enable efficient xylose fermentation and simultaneous in situ detoxification of acetic acid in plant cell wall hydrolysates. While the idea was innovative and has significant potential for the economic production of biofuels, the capacity of the engineered yeast strain to detoxify acetic acid into ethanol was insufficient for industrial applications. Therefore, we have optimized the acetate reducing pathway for enhanced detoxification of acetic acid into ethanol. Specifically, we modified the expression levels of key enzymes participating in the metabolic pathway and employed a mutant enzyme to overcome the allosteric regulation limiting metabolic fluxes. As a result, we were able to obtain the highest ethanol yield from xylose (0.463 g ethanol/g xylose) by engineered yeast. When a real plant cell wall hydrolysate was fermented by the optimized yeast, 18.4% more ethanol was produced than by an un-optimized strain. Furthermore, we have developed an engineered yeast strain that simultaneously converts all major carbon sources in plant cell wall hydrolysates—cellobiose, xylose, and acetic acid— into ethanol. Many desired phenotypes for producing cellulosic biofuels are often observed in industrial yeast strains. While most industrial yeast strains are polyploid so that it is difficult to use these strains for metabolic engineering applications, we demonstrated that industrial yeast strains can be engineered via haploid isolation for producing cellulosic biofuels. Our optimized yeast strain efficiently fermented a real plant cell wall hydrolysate for use in the industrial production of cellulosic biofuels.

Carbon dioxide fixation by engineered yeast during ethanol production from plant cell wall hydrolysates. Global climate change caused by the emission of carbon dioxide from the use of fossil fuels is a grand challenge to humanity. While production of biofuels is considered a carbon neutral-process, we have developed a carbon-negative process by introducing a synthetic carbon dioxide fixation pathway into a biofuel-producing yeast, achieving simultaneous lignocellulosic bioethanol production and carbon dioxide fixation. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were functionally expressed in an engineered yeast expressing bacterial chaperonins. The resulting strain was able to exhibit a higher yield of ethanol and reduced release of carbon dioxide during xylose fermentation. We are currently improving the capacity of carbon dioxide fixation in our engineered yeast through optimization of expression levels of key enzymes and reconfiguration of metabolic networks. This strategy has a great potential to alleviate greenhouse gas emissions during the production of next-generation biofuels.

Value-added biotransformation of food wastes. Most food wastes contain numerous organic compounds requiring use of costly waste treatment processes before discharging. For instance, whey is inevitably produced during the manufacturing of dairy products, such as cheese or cream. Recently, a huge amount of acid whey—whey containing organic acids—has been produced during the rapidly growing Greek yogurt industry.  As such, it is desirable to convert the fermentable sugar lactose in whey into value-added products instead of feeding animals or making biogas. To this end, we have developed engineered yeast strains capable of converting lactose into high-value products. In particular, we have built an engineered yeast strain capable of producing a functional sweetener (D-tagatose) from whey. Unlike existing technologies using multiple enzyme reactions and separation steps to produce tagatose from lactose, our engineered yeast can produce tagatose from lactose via one-step fermentation.

Precise genome editing in microorganisms for food and industrial applications. Many foods, such as cheese, wine, and bread, have been produced using microbial fermentation. Nonetheless, many issues have hindered the utilization of engineered microorganisms for fermented foods. Among the issues, safety concerns of genetic engineering procedures have remained because traditional genetic engineering methods employ antibiotic resistance genes and under-characterized genetic elements. Therefore, we have exploited emerging CRISPR/Cas9-based genome editing for precise and marker-less genetic manipulation of microorganisms which were previously not amenable for genetic engineering. For instance, it was difficult to introduce designed genetic perturbations into industrial polyploid yeast strains even though the polyploid yeast harbor beneficial traits for many applications. We demonstrated an effective strategy to precisely generate intended genetic perturbations in industrial polyploid yeast strains using the RNA-guided Cas9 nuclease system. In collaboration with Drs. Hans Blaschek and Ting Lu, we have made it possible to introduce designed genetic perturbations into Clostridium beijerinckii, a butanol-producing bacterium which was notoriously difficult to genetically modify.

Microbiome engineering using prebiotics and probiotics to promote gut health. We envision that metabolic engineering can be applied for producing prebiotics and engineering probiotic strains to promote human health. Human milk oligosaccharides (HMOs) are known to provide various health benefits for breast-fed infants. HMOs are also known to play important roles in reducing diarrheal diseases by preventing adhesion of pathogens and toxins to intestinal cells. Yet, chemically synthesized HMOs have been prohibitively expensive (~$100 per mg) for animal feeding experiments, let alone commercial applications. We have demonstrated the feasibility of producing HMOs by engineered microorganisms and filed a US patent disclosing scalable and economical production of HMOs by engineered microorganisms. We are also pioneering a radical idea of using engineered microorganisms to promote gut health. Using Cas9-based genome editing, we have made targeted genetic manipulations in Saccharomyces boulardii, a probiotic yeast which has been used for promoting gut health as well as preventing diarrheal diseases. We demonstrated the overexpression of a heterologous gene, the correct localization of a target protein, and the introduction of a heterologous metabolic pathway in the genome of S. boulardii. We are currently working on delivering bioactive molecules and therapeutic proteins into the gut using engineered S. boulardii for preventing and treating gastrointestinal diseases.

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